D27 myristic acid

In order to extract metabolites from the tissue, each sample was transferred to 2.0 ml ceramic bead mill tubes (bioExpress). Each sample received 450 ul of 90% cold methanol in diH2O for every 25 mg of tissue. The samples were then homogenized in an OMNI Bead Ruptor 24. Homogenized samples were then incubated at −20 °C for 1 hr. D4-succinic acid (Sigma 293075) was added to each sample as an internal standard. After incubation, all the samples were centrifuged at 20,000 x g for 10 min at 4 °C. 450 ul of supernatant was then transferred from each bead mill tube into a labeled, fresh micro centrifuge tube where another internal standard d27-myristic acid (CDN Isotopes: D-1711). Samples were then dried en vacuo. For metabolite extraction from serum, 90% methanol in diH2O containing D4-succinic acid was added to each sample to give a final methanol concentration of 80%. Samples were vortexed and incubated at −20 °C for 1 hr. After incubation, all samples were centrifuged at 20,000 x g for 10 min at 4 °C. Another internal standard, d27-myristic acid (CDN Isotopes: D-1711), was added to each sample. Process blanks were made using the extraction solvent and went through the same process steps as the real samples. The samples were then dried en vacuo.